The present invention relates to a method for detecting sequence-specific methylation in a biomolecule, comprising: (a) contacting a biomolecule with an
S-adenosyl-L-methionine-dependent methyltransferase in the presence of a detectable cofactor of said methyltransferase; and (b) detecting whether the recognition sequence of said methyltransferase has been modified with the cofactor or a derivative thereof, wherein modification of the recognition sequence of said methyltransferase is indicative of an absence of methylation at said recognition sequence. The present invention also relates to a cofactor which is specific for S-adenosyl-L-methionine-dependent methyltransferases, wherein said cofactor is an N-adenosylaziridine derivative with a reporter group attached to the 6 or 7 position of the adenine ring or attached to the aziridine ring. Moreover, the present invention relates to a complex of the cofactor of the present invention and a methyltransferase which normally uses S-adenosyl-L-methionine (AdoMet) as a cofactor. In addition, the present invention relates to a diagnostic composition comprising the cofactor of the present invention or the complex of the present invention. Finally, the present invention relates to the use of the cofactor of the present invention or the complex of the present invention for detecting sequence-specific methylation in DNA molecules.
Several documents are cited throughout the text of this specification. The disclosure content of the documents cited therein (including manufacture's specifications, instructions, etc.) is herewith incorporated by reference.
Any combination of steps (including single steps only) carried out in vitro and cited throughout this specification can also be carried out with cell extracts or in vivo.
The present invention is exemplified using DNA methylation found in humans. However, it can also be used to detect DNA methylation in other organisms as well as RNA methylation and protein methylation.
DNA methylation is found in almost all organisms (Jeltsch, (2002) ChemBioChem 3, 275-293). The DNA can contain the methylated nucleobases 5-methylcytosine (5-mCyt), N4-methylcytosine (4-mCyt) or N6-methyladenine (6-mAde) in addition to cytosine, adenine, thymine and guanine. These methylated nucleobases are formed by DNA methyltransferases (MTases) which catalyze the transfer of the activated methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the C5 carbon of cytosine, the N4 nitrogen of cytosine or the N6 nitrogen of adenine within their DNA recognition sequences (Cheng, (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 293-318). Since a particular nucleotide sequence may exist in its methylated or unmethylated form, DNA methylation can be regarded as an increase of the information content of DNA, which serves a wide variety of biological functions. In prokaryotes DNA methylation is involved in protection of the host genome from endogenous restriction endonucleases, DNA mismatch repair, regulation of gene expression and DNA replication. In eukaryotes DNA methylation plays a role in important regulatory processes such as gene silencing (Bird, (2002) Genes Dev. 16, 6-21, genomic imprinting (Feil and Khosla, (1999) Trends Genet. 15, 431-435), X-chromosome inactivation (Panning and Jaenisch, (1998) Cell 93, 305-308), silencing of intragenomic parasites (Yoder, (1997) Trends Genet. 13, 335-340), and carcinogenesis (Baylin, (1998) Adv. Cancer Res. 72, 141-196; Jones and Laird, (1999) Nat. Genet. 21, 163-167). Success of cancer treatments, in general, depends to a large extent on an early diagnosis of tumorgenesis. Therefore, there is an important need to develop early assays of general tumorgenesis.
It is considered that a number of particular forms of cancer are associated with changes in the regulation of gene expression. In many cases, the changes of gene expression can be traced back to an altered methylation pattern of chromosomal DNA. For a long time it was known that DNA methylation is a mechanism for altering gene expression without altering the coding function of a gene. The methylation reaction involves transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) in a cleft of the enzyme DNA (cytosine-5)-methyltransferase to form 5-methylcytosine (5-mCyt). Interestingly, 5-methylcytosines are not evenly distributed in the chromosomal DNA but tend to be located to CpG dinucleotides. The mammalian genome contains few isolated CpG dinucleotides which are largely methylated (Larsen, et al., (1992) Genomics 13, 1095-1107). More frequently observed are dinucleotide clusters of CpG's or “CpG islands” (Gardiner-Garden and Frommer, (1987) J. Mol. Biol. 196, 261-282) which are present in the promoter and exonic regions of approximately 40% of mammalian genes. CpG islands are areas of the genome, typically between 0.2 to about 1 kb in length, which are enriched in cytosines and guanines relative to the genome. CpG islands have been shown to be associated with the 5′ ends of all housekeeping genes and many tissue-specific genes, and with the 3′ ends of some tissue-specific genes. The 5′ CpG islands may extend through 5′-flanking DNA, exons and introns, whereas most of the 3′ CpG islands appear to be associated with exons. CpG islands are generally found in the same position relative to the transcription unit of equivalent genes in different species, with some notable exceptions (Gardiner-Garden and Frommer, (1987) J. Mol. Biol. 196, 261-82).
Methylation of cytosine residues contained within CpG islands of certain genes has been inversely correlated with gene activity. It is conceivable that this could lead to a decreased gene expression by a variety of mechanisms such as a local condensation of chromatin structure, inhibition of transcription factor-DNA binding, or by recruiting proteins which specifically interact with methylated CpGs indirectly preventing transcription factor binding. Increased methylation may also affect, e.g., the promoter region of tumor suppressor genes. Increased methylation in such regions may lead to progressive reduction of normal gene expression resulting in a population of cells having a selective growth advantage.
It is, however, not simply an increased methylation of DNA that is observed in cancer cells. Rather, distinct changes in the methylation pattern of DNA might be sufficient to alter gene expression in the cell and to induce carcinogenesis. In fact, it has been shown that cancer cells are associated with a characteristic pattern of CpG-methylation, distinct from the methylation pattern of their healthy progenitor cell. Hence, knowledge of the specific methylation pattern of chromosomal DNA of healthy and diseased cells can be exploited for developing markers that can be used for detection of diseases such as cancer.
Mapping of methylated regions in DNA (Rein et al., (1998) Nucleic Acids Res. 26, 2255-2264) has primarily relied on Southern hybridization approaches, based on the inability of methylation-sensitive restriction enzymes to cleave sequences which contain one or more methylated CpG sequences. This method provides for an assessment of the overall methylated status of CpG islands, including some quantitative analysis, but is relatively insensitive, requires large amounts of sample and can only provide information about those CpG sequences found within sequences recognized by methylation-sensitive restriction enzymes.
A more sensitive approach is based on the detection of methylated patterns by using a combination of methylation sensitive enzymes and the polymerase chain reaction (PCR). After digestion of DNA with the enzyme. PCR will amplify from primers flanking the restriction sequence only if DNA cleavage was prevented by methylation. Like Southern-based approaches, this method can only monitor CpG-methylation in methylation-sensitive restriction sites. Another method that avoids the use of restriction endonucleases utilizes bisulfite treatment of DNA to convert all unmethylated cytosines to uracils. The altered DNA is amplified and sequenced to show the methylation status of all CpG sequences. Alternatively, the amplified DNA can be analyzed in hybridization experiments.
The major disadvantage of the approaches of the prior art is that the primary modification of native DNA does not directly lead to incorporation of a detectable label which strongly restricts applicable methods for the following detection step.
Recently, a novel approach for sequence-specific labelling of DNA using a newly designed fluorescent cofactor for the DNA (adenine-6)-methyltransferase from Thermus aquaticus (M.TaqI) has been presented (Pljevaljcic et al., (2003) J. Am. Chem. Soc. 125, 3486-3492). Naturally, M.TaqI catalyze the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5′-TCGA-3′ DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) leading to sequence- and base-specific methyl group transfer. Most importantly, M.TaqI, like other DNA methyltransferases (MTases) can only transfer one methyl group to its target base and DNA with a fully methylated recognition sequence is not further modified.
Replacement of the methionine side chain of the natural cofactor S-adenosyl-L-methionine (AdoMet) by an aziridinyl residue leads to M.TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to the target adenine in DNA. The adenosyl moiety is the molecular anchor for cofactor binding. Attachment of a fluorophore via a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. The newly designed cofactor, 8-amino[1″-(N″-dansyl)-4″-aminobutyl]-5′-(1-aziridinyl)-5′-deoxyadenosine, can be used to sequence-specifically label DNA in a M.TaqI-catalyzed reaction.
It would be desirable to use the newly designed fluorescent cofactor for labelling reactions catalyzed by DNA (cytosine-5)-methyltransferases with a recognition sequence embracing the CpG motif of CpG islands. Since methylation of cytosine residues should block enzymatic coupling of the labelled cofactor, a successful labelling reaction would be indicative of a non-methylated CpG sequence, whereas a failure to couple a labelled cofactor would be indicative of a methylated CpG sequence. Thus, this reaction could be used to monitor the methylation status of chromosomal DNA.
Haemophilus haemolyticus naturally produces a DNA (cytosine-5)-methyltransferase, M.HhaI, with the required specificity and methylates the first cytosine within the double-stranded DNA sequence 5′-GCGC-3′. However, no detectable N-adenosyl-aziridine cofactors or derivatives of M.HhaI are available in the art. Therefore, the technical problem underlying the teaching of the present application was to provide a detectable cofactor for M.HhaI and other S-adenosyl-L-methionine-dependent methyltransferases and methods for detecting alterations in the methylation status of DNA.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly the present invention relates to a method for detecting sequence-specific methylation in a biomolecule, comprising: (a) contacting a biomolecule with an S-adenosyl-L-methionine-dependent methyltransferase in the presence of a detectable cofactor of said methyltransferase; and (b) detecting whether the recognition sequence of said methyltransferase has been modified with the cofactor or a derivative thereof, wherein modification of the recognition sequence of said methyltransferase is indicative of an absence of methylation at said recognition sequence, wherein the N-adenosylaziridine derivative is represented by formula (I),
whereinW is selected from N and CH,X is N or CR1,Y is NH2 or NHR2,Z is H, R3 or CH2CH(COOH)(NH2),with the proviso thatif X is CR1, Y is NH2 and Z is H or CH2CH(COOH)(NH2),if X is N and Y is NHR2, Z is H or CH2CH(COOH)(NH2),if X is N and Y is NH2, Z is R3,R1 is selected from —(CH2)nR4, —(CH═CH)m(CH2)nR4, —(CH2)o(CH═CH)m(CH2)nR4, —(C≡C)m(CH2)nR4, —(C)m(C6H4)o(CH2)nR4, —(C6H4)m(CH2)nR4, —CO(CH2)nR4 and —S(CH2)nR4;R2 is selected from —(CH2)nR4, —(C6H4)m(CH2)nR4, —CO(C6H4)m(CH2)nR4 and —CO(CH2)nR4;R3 is selected from —(CH2)nR4, —(CH═CH)m(CH2)nR4, —(C≡C)m(CH2)nR4, —(C6H4)m(CH2)nR4 and —CONH(CH2)nR4;R4 is selected from —NHR5, —NHCO(CH2)pSR5, —SR5, —OR5, —O(C2H5O)n(C2H5)NHR5, —CH2NHNHR5, —NHCOCH(CH2SH)NHR5 and —CONHR5;R5 is selected from fluorophores, affinity tags, crosslinking agents, chromophors, proteins, peptides, amino acids which may optionally be modified, nucleotides, nucleosides, nucleic acids, carbohydrates, lipids, PEG, transfection reagents, beads, intercalating agents, nucleic acid cleaving reagents and nanoparticles (e.g. gold cluster) andn, m, o and p are independently selected from 0 or an integer from 1 to 5000.
The term “detecting sequence specific methylation”, as used throughout the invention, means to assess whether the acceptor site within the recognition sequence of a methyltransferase (MTase) is modified by the addition of a methyl group. Preferably, said acceptor site is part of the recognition sequence of a DNA methyltransferase. More preferably, said DNA methyltransferase is selected from M.HhaI and M.TaqI, M.BseCI and M. SssI. When the DNA methyltransferase is M.HhaI, it is preferred that the acceptor site is cytosine within the 5′-CG-3′ sequence which is embedded into the larger 5′-GCGC-3′ recognition sequence of M.HhaI.
The term “biomolecule” means DNA, RNA or (poly)peptide. The term “(poly)peptide” refers alternatively to peptide or to polypeptide. Peptides conventionally are covalently linked amino acids of up to 30 residues, whereas polypeptides (also referred to as “proteins”) comprise 31 and more amino acid residues. Preferably, the biomolecule is chromosomal or genomic DNA.
The term “contacting a biomolecule with a methyltransferase” means bringing into contact the biomolecule with the methyltransferase. Generally, this may be done by adding the methyltransferase to a sample containing the biomolecule. Alternatively, the sample containing the biomolecule may be added to a solution containing the methyltransferase. The skilled person knows that particular buffer conditions might be required for optimal enzyme activity. These conditions are either known to the skilled person or can be obtained by studying enzyme activity under various assay conditions.